Mycobacteria are a genus of the phylum Actinobacteria that includes the human pathogens M. tuberculosis and M. leprae and their avirulent relative M. smegmatis. Human infections with M. tuberculosis (the agent of tuberculosis) and M. leprae (agent of leprosy) cause substantial human suffering. Tuberculosis accounts for ~2 million deaths annually. M. tuberculosis is increasingly antibiotic resistant, solely through the acquisition of mutations in chromosomal genes. The importance of mutagenesis in the evolution of M. tuberculosis antimicrobial resistance, and the related importance of DNA repair pathways in resisting host-inflicted DNA damage, prompts our interest in the mechanisms by which mycobacteria respond to and repair DNA damage. In addition to this important relevance to human health, mycobacteria have emerged as fertile model system to study prokaryotic DNA repair, due to the complexity of the pathways involved and the novel mechanisms that govern these pathways. Our long range goals in this project are to elucidate the DNA repair mechanisms of Mycobacteria. Our work supported by this award has shown that mycobacterial DNA repair differs from the classic E. coli model system with respect to the number of pathway options for the repair of DNA double-strand breaks (DSBs), the roster of DNA repair enzymes, and the regulation of the DNA damage response (DDR). Our premise is that understanding the distinctive features of mycobacterial DNA repair will illuminate the evolution and diversification of repair strategies and suggest new approaches to combat mycobacterial infection and emergence of antibiotic resistance. Our agenda for the next phase of the project focuses on three themes in mycobacterial DNA repair: (i) the mechanism of DSB resection during homologous recombination (HR) by the AdnAB helicase-nuclease; (ii) the role of RecA phosphorylation and RecA interaction with membrane phospholipids in controlling the DNA damage response; and (iii) the structure and repair activities of the DNA helicase Lhr. We propose a combined approach that leverages integrated biochemical, structural, and genetic approaches to understand these DNA repair systems of mycobacteria. Through these studies we will elucidate new mechanisms of DNA repair in mycobacteria, discoveries that will both advance basic knowledge of prokaryotic DNA repair and elucidate pathways relevant to M. tuberculosis drug resistance and pathogenesis.